Pesticide induced immunotoxicity in humans: a comprehensive review of the existing evidence.

The immune system can be the target of many chemicals, with potentially severe adverse effects on the host's health. In Western countries pesticides, together with new and modified patterns of exposure to chemicals, have been implicated in the increasing prevalence of diseases associated with alterations of the immune response, such as hypersensitivity reactions, certain autoimmune diseases and cancers. Xenobiotics may initiate, facilitate or exacerbate pathological immune processes, resulting in immunotoxicity by induction of mutations in genes coding for immunoregulatory factors, modifying immune tolerance and activation pathways. The purpose of this article is to update the evidence of pesticide immunotoxicity. Even if experimental data as well as sporadic human studies indicate that some pesticides can affect the immune system, overall, existing epidemiological studies are inadequate to raise conclusions on the immunotoxic risk associated to pesticide exposure. The available studies on the effects of pesticides on human immune system have several limitations including poor indication on exposure levels, multiple chemical exposures, heterogeneity of the approach, and difficulty in giving a prognostic significance to the slight changes often observed. Further studies are necessary, and they should be preferably carried out through comparison of pre and post-exposure findings in the same group of subjects with a matched control group. Attempt should be made to define the prognostic significance of slight changes often observed. Animal and in vitro studies are also important and necessary to scientifically support epidemiological evidences on pesticide-induced immunotoxicity.

Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens.

Several European Union legislations request the use of in vitro methods for toxicological evaluations, including sensitization, in order to increase consumer safety but also to reduce the use of animals. The EU project SENS-IT-IV addresses the need of developing predictive in vitro tests to assess contact and respiratory hypersensitivity reactions. In this context, we have recently reported the possibility to use IL-18 production in the human keratinocyte cell line NCTC 2544 to discriminate contact sensitizer from irritants and low molecular weight respiratory allergens. The aims of the present study were to further develop this assay in order to optimize experimental conditions; to develop a 96-well plate format to establish a high throughput assay; to test the performance of other available keratinocyte cell lines, and to understand the signal transduction pathway involved in p-phenylenediamine (PPD)-induced IL-18 production. If cells reach confluence at the moment of treatment, the ability to identify contact allergens is lost; therefore a careful check for the optimal cell density using PPD as reference contact allergen is critical. In our hands, a cell density of 1-2.5 × 10(5)cells/ml gave optimal stimulation. In order to develop a high throughput test, cells seeded in 96-well plate were exposed to contact allergens (2,4-dinitrochlorobenzene, p-phenylenediamine, isoeugenol, cinnamaldehyde, tetramethylthiuram disulfite, resorcinol, cinnamic alcohol and eugenol), irritants (phenol, sodium laurel sulphate, lactic acid and salicylic acid) and respiratory allergens (hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride). A selective increase in total (intracellular plus released) IL-18 was observed 24h later in cells treated with contact allergens, whereas no changes were observed following treatment with respiratory allergens and irritants, confirming previous results obtained in a 24-well format assay. A selective induction of IL-18 was also obtained testing with PPD other keratinocyte cell lines, namely HPKII and HaCaT, with the HPKII showing the highest stimulation index. Regarding the signal transduction pathway, we could demonstrate using selective inhibitors a role for oxidative stress, NF-κB and p38 MAPK activation in PPD-induced IL-18 production. In conclusion, results obtained suggest that the production of IL-18 represents a promising endpoint for the screening of potential contact allergens. The assay can be performed in a 96-well plate format, different keratinocyte cell lines can be used, and a role for oxidative stress in contact allergen-induced IL-18 was demonstrated.

Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro.

The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alternatives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency. We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency. THP-1 cells were exposed to the contact allergens (p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5-15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release. A significant correlation between IL-8 release and the LLNA EC(3) was found (Pearson correlation r=0.743, p=0.0036, n=12). On the contrary, the activation of p38 MAPK showed no significant correlation between LLNA data and vigor of p38 MAPK activation. Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification.

Molecular mechanism of teratogenic effects induced by the fungicide triadimefon: study of the expression of TGF-beta mRNA and TGF-beta and CRABPI proteins during rat in vitro development.

Azole derivatives are teratogenic in rats and mice in vitro and in vivo. The postulated mechanism for the dysmorphogenetic effects is the inhibition of retinoic acid (RA)-degrading enzyme CYP26. Azole-related abnormalities are confined to structures controlled by RA, especially the neural crest cells, hindbrain, cranial nerves, and craniofacial structures, through a complex signal cascade. The aim of this work is to study the expression of signal molecules activated by RA (TGF-betas) or involved in the modulation of cellular RA concentrations (CRABPI). E9.5 (9.5 day post coitum old embryos) rat embryos, exposed in vitro to triadimefon (FON) for 24 h, were examined or cultured in normal serum for extra 4, 16, and 24 h. RT-PCR was performed to quantify TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII, and TGF-betaRIII mRNA in the hindbrain after 24 h of culture. TGF-beta1, TGF-beta2, and TGF-betaRI were found significantly decreased by FON exposure, and consequently their protein expression was analyzed by Western blot and immunohistochemistry. In both controls and FON-exposed embryos, TGF-beta1 and TGF-betaRI were detected at 24 and 24+4 h; TGF-beta2 was present only at 24 h. Only TGF-beta1 was expressed at the level of hindbrain and branchial tissues. After quantization, TGF-beta1 was reduced in the FON group. The expression of CRABPI was observed at all developmental stages. However, in FON-exposed embryos, it was increased at 24 and 24+4 h. The hindbrain distribution of CRABPI-positive cells was abnormal in FON-exposed embryos. The results show that the two RA-related molecules (TGF-beta1 and CRABPI) are altered by FON exposure in vitro.

Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.

BACKGROUND: Skin immunosenescence accounts for increased susceptibility in the elderly to cutaneous infections and malignancies, and decreased contact hypersensitivity and response to vaccination. We have recently shown in immune cells that decreased expression of the receptor for activated C kinase (RACK)-1 underlies defective protein kinase C (PKC) activation and functional immune impairment with ageing. OBJECTIVES: This study was designed to determine if an age-related decline in skin RACK-1 expression was present and whether it correlated with defective tumour necrosis factor (TNF)-alpha production. METHODS: PKC isoforms and RACK-1 expression were evaluated by Western blot analysis and by immunofluorescence in skin obtained from Sprague-Dawley rats of different ages. TNF-alpha release by epidermal cells induced by lipopolysaccharide, 12-O-tetradecanoyl-phorbol-13-acetate and the contact allergen dinitrochlorobenzene was assessed by the L929 biological assay. RESULTS: Skin obtained from old rats (> 18 months) showed decreased RACK-1 immunoreactivity if compared with young rats (< 3 months). RACK-1 preferentially interacts with PKC beta. Despite a similar total skin content of this isoform, the reduced expression of RACK-1 was associated with a decreased translocation of PKC beta in the membrane compartment. The defective PKC beta translocation associated with ageing correlated with decreased TNF-alpha release from epidermal cells following treatment with different inflammatory stimuli. CONCLUSIONS: Overall, we demonstrated for the first time a decrease in RACK-1 expression, defective PKC beta translocation and reduced TNF-alpha release in epidermal cells with ageing. These alterations might be mechanistically significant, and provide a new understanding of the consequences of ageing on skin immunology.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route. The physicochemical characteristics of the chemical and the pattern of cosmetic use would affect the long-term average internal dose that is compared with the relevant TTC value. Analysis has shown that the oral TTC values are valid for topical exposures and that the relationship between the external topical dose and the internal dose can be taken into account by conservative default adjustment factors. The TTC approach relates to systemic effects, and use of the proposed procedure would not provide an assessment of any local effects at the site of application. Overall the TTC approach provides a useful additional tool for the safety evaluation of cosmetic ingredients and impurities of known chemical structure in the absence of chemical-specific toxicology data.

Low level exposure to chemicals and immune system.

Industrialized countries are facing an increase of diseases attributable to an alteration of the immune system function, and concern is growing that this trend could be at least partially attributable to new and modified patterns of exposure to chemicals. Among chemicals matter of concern, pesticides can be included. The Authors have reviewed the existing evidence of pesticide immunotoxicity in humans, showing that existing data are inadequate to raise conclusions on the immunotoxic risk related to these compounds. The limits of existing studies are: poor knowledge on exposure levels, heterogeneity of the approach, and difficulty in giving a prognostic significance to the slight changes often observed. To overcome these limits, the Authors have proposed a tier approach, based on three steps: the first, addressed at pointing out a possible immunomodulation; the second, at refining the results and the third one, when needed, to finalize the study and to point out concordance with previous results. Studies should preferably be carried out through comparison of pre- and post-exposure findings in the same groups of subjects to be examined immediately after the end of the exposure. A simplification of the first step approach can be used by the occupational health physician and the occupational toxicologist. Conclusions on the prognostic significance of the slight changes often observed will be reached only by validating the hypothesis generated by field studies with an epidemiological approach. In this field, the most useful option is represented by longitudinal perspective studies.

Ochratoxin A in conventional and organic cereal derivatives: a survey of the Italian market, 2001-02.

Ochratoxin A is a mycotoxin produced mainly by Penicillium verrucosum and Aspergillus ochraceus. Although typically considered a cereal contaminant, it has also been detected in dried fruit, nuts, meat and derivatives. To estimate the quantity of ochratoxin A that might be ingested by Italian consumers from these foods, 211 cereal derivatives (flours and bakery products) were analysed by high-performance liquid chromatography. Products were from conventional and organic agriculture and from integrated pest management agriculture. All commercial flours and derivatives examined contained ochratoxin A at concentrations very much below the legal limit (3 microg kg(-1)): the highest value, 0.816 microg kg(-1), was detected in a sample of spelt whole flour from organic agriculture. In many samples, the ochratoxin content was below the limit of detection; only rarely did values exceed 0.5 microg kg(-1). In baby foods, four samples were above the particularly restrictive Italian legal limit of 0.5 microg kg(-1). Although some significant differences were found between samples from conventional and organic agriculture when some product categories were examined (namely, baby foods as semolina and rice creams), no important difference was found between the two types of agricultural practice when all types of cereal derivatives were considered together.

Analysis by high-performance liquid chromatography of teucrin A in beverages flavoured with an extract of Teucrium chamaedrys L.

Due to its liver toxicity, the medicinal use of germander (Teucrium chamaedrys L.) was banned in some countries. Nevertheless, alcoholic extracts are still permitted as flavour ingredients since they are fundamental in providing a bitter aromatic taste. Teucrin A represents the substance of major concern regarding the potential toxicity of germander. Hence, teucrin A represents the best analytical and toxicological marker of alcoholic extracts of T. chamaedrys. A sensitive high-performance liquid chromatography method to detect teucrin A in beverages is reported. Teucrin A was prepared by isolation from the plant extract using column chromatography and crystallization. The identity and purity (99%) were established by melting point, nuclear magnetic resonance and liquid chromatography-mass spectrometry. The high-performance liquid chromatography procedure was validated and its intra- and interday performance was established (relative standard deviation < or = 13% and error < or = 10%). In-house validation was carried out by analysing samples of beverages not containing T. chamaedrys spiked with a range of concentrations of teucrin A. The limit of detection was 0.1 ppm and the limit of quantification was 0.3 ppm. Teucrin A accounted for about 70% of the neo-clerodane diterpenoids found in the total extract of a specimen of T. chamaedrys. The content (+/- standard deviation) in 18 batches of different geographical origin was 2338 +/- 740 ppm, per cent coefficient of variation = 32, minimum-maximum = 999 - 3445 ppm. The mean level of teucrin A in 10 bottles of the same brand was 6.1 +/- 0.8 ppm, per cent coefficient of variation = 12. In 10 different brands found on the Italian market, the content of teucrin A ranged from not detectable to 10 ppm.

Resistance to silica-induced lung fibrosis in senescent rats: role of alveolar macrophages and tumor necrosis factor-alpha (TNF).

The purpose of the present study was to investigate the effect of aging on silica-induced lung toxicity. In young animals silica induced a significant increase in bronchoalveolar lavage tumor necrosis factor-alpha (TNF), lactate dehydrogenase as well as in cell numbers, which correlate with increased collagen deposition and silicotic nodules formations. In old rats, however, no changes in bronchoalveolar lavage or lung parameters were observed following silica instillation. These in vivo results were also confirmed in vitro, where silica failed to induce TNF release in alveolar macrophages obtained from old animals. This defective response to silica could be explained with defective protein kinase C translocation, due to a reduction in its anchoring protein RACK-1 with aging. Overall, these data indicate that the understanding of the molecular mechanisms undelaying toxicity is crucial to define the influence of age on the toxic response and progression of the disease.

[Assessment of cumulative risk of mixtures with multiple target organs]. / Valutazione del rischio cumulativo derivante da miscele con effetto su differenti bersagli.

Humans are exposed to very complex environmental mixtures. In contrast to this reality, most laboratory investigations of mixtures, with exception of mutagenicity assays, have focused on mixtures comprised of a few chemicals. Component-based approaches that use information on the individual chemical contained in the mixture are not by themselves sufficient for complex, environmental mixtures because significant portions of the mixtures mass are unidentified. Assessment of the cumulative risk posed by exposure to multiple chemicals is a problem the USEPA's and FDA's program discuss regularly. In USA the Food Quality Protection Act of 1996 directs the Office of Pesticide Programs to include in its assessments the risk associated with the cumulative effects of pesticides that have a common mode of action. organophosphorus pesticides (OPs) have been the first class addressed, based on the common mechanism of acetylcholinesterase inhibition. Interestingly toxicity studies focussing on component-based analysis of mixtures indicated that for chemicals not sharing the same target and mechanism, the type of combined action or interaction found when each chemical in the mixture was present at clearly toxic effect levels did not predict the response observed when each chemical was present at or below the lowest observed adverse effect level (LOAEL). Moreover, mixtures of chemicals produced toxicity only for those chemicals that showed the same toxic effect on the same target organ when given singly at dose above the LOAEL values. However, end-points chosen in routine toxicity studies do not offer a wide dynamic range of testable, significant responses around and above the NOAEL. So far sensitive methods that detect low level changes have not yet been employed in mixtures testing protocol. Toxicogenomics integrated functional genomics with classical toxicology and we believe that it has great potential to revolutionize mixtures research.

Evaluation of residual immunoreactivity in red and white wines clarified with gluten or gluten derivatives.

Gluten or hydrolyzed gluten could be a suitable alternative to animal proteins in the wine clarification process, but their residues could represent a risk for individuals suffering from coeliac disease or allergic to cereal proteins. The aim of this study was to investigate the presence of gluten in wines treated with gluten or its hydrolysate in the clarification process and to assess its antigenicity in commercial products. The presence of residual immunoreactive gluten was evaluated by electrophoresis (SDS-PAGE) and immunoblotting. Data obtained in several red and white wine samples showed that no residue was detectable in any of the red wines. In white wines, gluten reduced the protein content less completely, but most samples showed no immunoreactivity after the wine had been treated with gluten or its derivatives, either alone or combined with bentonite, silica gel or tannins. The use of gluten derivatives coupled with bentonite was the most effective method of removing immunoreactive protein in white wines. In conclusion, the use of gluten derivatives in wine clarification seems to exclude a risk for subjects susceptible to coeliac disease or gluten allergy. However, it is recommended that wine producers continuously monitor the clarification process in order to protect the most sensitive individuals.

Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases.

Interleukin (IL)-1beta is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1beta and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1beta affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1beta (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of approximately 45%. This effect occurred only when neurons were pretreated with IL-1beta, whereas it was absent if IL-1beta and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1beta was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 microm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1beta. Finally, 0.05 ng/ml IL-1beta increased by approximately 30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1beta increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.

Evaluation by SDS-Page and immunoblotting of residual antigenicity in gluten-treated wine: a preliminary study.

Hydrolyzed gluten could be a suitable alternative to animal proteins in the wine clarification process, but the residual proteins could constitute a risk for subjects suffering from celiac disease or allergy to cereals. The aim of this study was to investigate possible traces of gluten in treated wine and to assess its antigenicity in commercial products. The presence of gluten in treated wine was evaluated by an electrophoretic method [sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] and its immunoreactivity was evaluated by immunoblotting. No traces of protein were found in untreated samples. A small quantity of protein was detected in treated wine but this produced no significant immunochemical reaction. In an experimental clarification process, a protein fraction was detectable in untreated samples and in the first stages of the clarification process. However, there was no significant gluten-associated immunochemical reaction in clarified wine samples, confirming strong binding between the clarifying agent and the phenolic fraction. In conclusion, the clarifying process strongly reduced the amount of protein material, at least in red wines. Under the most restrictive tests of the presence of gluten in the product, the predictable residue of gluten in wine was safe for celiac subjects. For allergic subjects the data are less conclusive because there is no known limit for allergic reactions, but clear labeling of the method of treating the wine should also protect this group of consumers.

Use of differential display-polymerase chain reaction to identify genes selectively modulated by chemical allergens in reconstituted human epidermis.

In the screening of topical drugs, cosmetics and other chemicals for human use, it is very important, both from a safety and an economic point of view, to have biological markers to discriminate irritant and allergic contact dermatitis that have different impacts on human health. Owing to their anatomical location, keratinocytes are among the first cells to be exposed to various antigens and the use of these cells as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for cutaneous application is amply justified. The purpose of this work was to identify new genes selectively modulated by skin toxicants. Commercially available reconstituted human epidermis (Epiderm) was treated for 18 h with sodium dodecyl sulfate (SDS) 0.4 mg/ml, as reference irritant, or with dinitrochlorobenzene (DNCB) 0.2 mg/ml, as reference allergen, or with vehicle control. Differential display PCR (DD-PCR) was performed. Results identified adipose differentiation-related protein (ADRP) as up-regulated by both irritant and allergen, and KIAA0368 as selectively up-regulated by contact allergen. These data indicate the enormous potential of functional genomic techniques, which allow the identification of genes not immediately connected with the immune response, or even novel genes with unknown functions, which nevertheless may be potential markers of skin irritation and allergy.

Hazard identification by methods of animal-based toxicology.

This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.

Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation.

Ochratoxin A is a typical cereal contaminant with strong nephrotoxic activity. To estimate the quantity of ochratoxin A that can be taken in by a child in the weaning period, several samples of cereal-based baby foods were analysed. Although most samples analysed contained ochratoxin A in undetectable amounts or below the Italian legal limit of 0.5 microg kg(-1), some irregular products were found. In particular, the analyses of the 119 batches (338 samples) of baby foods considered indicated that: 20 batches (16.8%) contained detectable quantities of ochratoxin A and four of these (3.4% of the total) contained ochratoxin A above the Italian permitted value. All samples coming from agricultural practices based on integrated pest management contained undetectable amounts of ochratoxin A, while approximately 5% of batches coming from conventional and organic agricultural practices were above the legal limit. On the basis of the established provisional tolerable weekly intake (PTWI), there is no significant toxicological risk for a child who occasionally consumes a formula with ochratoxin concentration slightly above the permitted level. However, stricter controls have to be applied to reject the batches containing irregular concentrations of ochratoxin A.

Dehydroepiandrosterone and the relationship with aging and memory: a possible link with protein kinase C functional machinery.

A progressive decline of cognitive and memory functions, compared to the average young-life performance, characterizes brain aging. The changes in performance may depend upon altered activity of neurotransmitters acting on attention and memory trace formation (acetylcholine, catecholamines, glutamate, for example) or the failure of the transduction mechanisms linked to receptor activation. One of the fundamental cellular changes associated with brain aging is the alteration of mechanisms involving the activity of the calcium-phospholipid-dependent protein kinase C (PKC). A crucial event for the activation of protein kinase C is its translocation from the cytosol to different intracellular sites and recent studies have demonstrated the key role played by several anchoring proteins in this mechanism. The defective activation of PKC-dependent pathways during aging is due to a defective mechanism of translocation of the kinase because of reduced levels of the major anchoring protein RACK-1 (receptor for activated C kinase). Pharmacological strategies aimed at the correction of age-associated memory deficits have been mostly focused on neurotransmitters using direct or indirect agonists. More recently, attention has been paid to the memory enhancing properties of some steroid hormones, namely 'neurosteroids'. Among these the activities of dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulfates, have been extensively studied. These neuroactive steroids, can regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. We addressed the possibility that DHEA, among other neurosteroids, could modulate directly the age-associated impairment of PKC signal transduction and provide experimental evidence that DHEA can revert the alteration of RACK-1 anchoring protein expression.

Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro.

Human immunodeficiency virus infection is often followed by neurodegeneration, the cause of motor and cognitive impairment in some patients affected by acquired immunodeficiency. Several in vitro data indicate glycoprotein (gp) 120 as one of the substances responsible for the neurodegenerative event that takes place only if non-neuronal cells (glial cells) are present. Our purpose was to investigate the molecular mechanisms through which glial cells could affect neuron viability after exposure to gp120 protein. We used a sandwich co-culture of primary hippocampal neurons and primary glial cells, where the two cell populations face each other but are separable. Exposure of 1-week-old rat hippocampal neurons in co-culture with glia to 600 pM gp120 protein resulted in the death of 30% of neurons after 6 days of treatment. A significant increase of intracellular calcium ([Ca2+]i), evident 72 h after gp120 exposure (control 45.8+/-7.6 nM, gp120 176.5+/-43.6 nM), preceded neuron death. The gp120 protein affected neither the viability nor the morphology or [Ca2+]i of glial cells. However, a significant amount of reactive oxygen species as well as of interleukin-1beta was produced. Treatment of the co-culture with an antibody against interleukin-1beta prevented neuron increase of [Ca2+]i and cell death but not glial production of reactive oxygen species, whereas prior incubation of glial cells with Trolox, an antioxidant analog of vitamin E, down-regulated interleukin-1beta expression and completely prevented neuron cell death. Our results indicate that reactive oxygen species produced in glial cells by gp120 exposure cause neurodegeneration by inducing the synthesis of interleukin-1beta.

Antigenic determinants of bovine serum albumin.

BACKGROUND: Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic characteristics have been described in several papers. The aim of this research was the identification of the BSA antigenic determinants. METHODS: This study was performed using limited proteolysis and an immunoblotting technique, in which a commercial murine antibody and sera from children sensitized to BSA were used. RESULTS: Findings suggest amino acids (aa) 524-598 as an epitopic area for human species. The most critical sequence seems to be aa 524-542, even if it must be included in a longer fragment to be recognized by antibodies. Murine IgG antibodies also recognize fragments contained in the first half (NH(2)-terminal portion) of BSA. CONCLUSIONS: The results presented in this study indicate that the epitopic sites of an antigenic protein can be different when different species are considered, so that data obtained with antibodies from animal species cannot be directly extrapolated to the behavior of human IgEs.